Further, Panitumumab was able to switch the MAPK activation in WITT cells. Open in a separate window Figure 1 Western Blot analysis for the evaluation of inhibition of Trametinib and Panitumumab UAMC 00039 dihydrochloride targetsCell lines were treated with 50 nM Trametinib (Tram) and 5 M Panitumumab (Pan) in monotherapy or in combination (Combo) and the expression of p-MAPK, MAPK, p-EGFR, EGFR and Vinculin was investigated. Trametinib slows tumor growth and inhibits angiogenesis in xenograft models of K-RAS mutated BTC Preclinical activity of Trametinib and Panitumumab was also evaluated in EGI-1, MT-CHC01 and WITT xenografts; 5106 cells were subcutaneously injected in the right flank of 28 mice and four groups (n=7) were created. cells. While Trametinib inhibited cell growth in K-RAS mutated UAMC 00039 dihydrochloride cell lines, Panitumumab had no effect on proliferation independently by K-RAS status. The addition of Panitumumab to Trametinib did not significantly potentiate its anti-proliferative effect also in mutated cells. growth of K-RAS mutated BTC cell lines The preclinical activity of the anti-EGFR Panitumumab, of the MEK inhibitor Trametinib, and of their combination was assessed using seven BTC cell lines expressing basal level of the targets and with different K-RAS mutational status. Two cell lines, the ICC cell line MT-CHC01 and the extrahepatic cholangiocarcinoma (ECC) cell line EGI-1 are mutated for K-RAS (G12D); the (ECC) WITT and TFK-1 cells, the gallbladder carcinoma (GBC) TGBC1 cells, the (ICC) HUH28 and the ICC mixed to hepatocarcinoma KMCH cells were K-RAS WT. IC50 values showed that this K-RAS mutated cell lines were sensitive to Trametinib, with an IC50 of 3.12 and 6.25 nM, respectively, while the other cells were unresponsive. All the cell lines were insensitive to Panitumumab (IC50 5 M) independently from the K-RAS status (Table ?(Table1).1). The combination did not potentiate the effect of Trametinib EGF alone in any cell lines (data not shown). Table 1 IC50 values of drugs in BTC cell lines with different K-RAS genomic status and models, the three tumorigenic cell lines EGI-1, WITT and MTCHC01. Cell lines were treated with 50 nM of Trametinib, 5 M of Panitumumab, or their combination for 3 hours. Western blot analysis (Physique ?(Determine1)1) demonstrated that Trametinib was able to switch off the MAPK1,2 activation in all the cell lines, independently by K-RAS status. It is interesting to note that in EGI-1 cells, Trametinib was also able to inhibit EGFR phosphorylation and, even less evident, also in WITT cells. Panitumumab reduced phospho-EGFR expression in EGI-1 cells and slightly in WITT cells. Further, Panitumumab was able to switch the MAPK activation in WITT cells. Open in a separate window Physique 1 Western Blot analysis for the evaluation of inhibition of Trametinib and Panitumumab targetsCell lines were treated with 50 nM UAMC 00039 dihydrochloride Trametinib (Tram) and 5 M Panitumumab (Pan) in monotherapy or in combination (Combo) and the expression of p-MAPK, MAPK, p-EGFR, EGFR and Vinculin was investigated. Trametinib slows tumor growth and inhibits angiogenesis in xenograft models of K-RAS mutated BTC Preclinical activity of Trametinib and Panitumumab was also evaluated in EGI-1, MT-CHC01 and WITT xenografts; 5106 cells were subcutaneously injected in the right flank of 28 mice and four groups (n=7) were created. After two/three weeks, tumors volume reached 100-200 mm3. Mice were then randomized to receive different treatments: the first cohort was intraperitoneally treated with Panitumumab (200g/mouse twice a week), the second cohort orally received (by gavage) Trametinib (0.3 mg/kg/die), another cohort received both drugs, and the last cohort was treated with the drug diluents as a control. Treatment was stopped at the day 28 for MT-CHC01 for their aggressiveness, while for the other two xenografts, treatment was continued up to 35 days. Tumors were calibrated weekly. One day after the last drug administration, mice were sacrificed and tumors were UAMC 00039 dihydrochloride harvested; curves of tumor volumes showed that in xenografts harboring K-RAS mutation, in particular in the EGI-1 xenografts, Trametinib drastically slowed the tumor growth down (p 0.0001) (Physique ?(Physique2A2A and ?and2D)2D) compared to the control arm. In EGI-1, Panitumumab did not significantly potentiate the effectiveness of Trametinib, which appears to be the real player model (Physique ?(Physique2B2B and ?and2E).2E). In K-RAS WT WITT xenografts, only the drug combination slowed the tumor growth (p=0.01) (Physique ?(Physique2C2C and ?and2F2F). Open in a separate window Physique 2 anti-tumor activity of Trametinib and Panitumumab and their combination in UAMC 00039 dihydrochloride human BTC preclinical modelsThe graphs indicate the median tumor volume (mm3) weekly measured (Panel A: EGI-1; Panel B: MT-CHC01; Panel C: WITT): 0 (start of treatment), 7, 14, 21, 28 and 35 days after treatment with Trametinib (Tram 0.3 mg/kg/die), Panitumumab (Pan 200g/mouse/twice a week), their combination (Combo), or drug vehicles (NT) (error bars: SD). Seven mice for each arm of treatment in three impartial experiments were used. Panel D, E and F: representative tumors derived from EGI-1, MT-CHC01 and WITT xenografts, respectively. Effect of Trametinib and/or Panitumumab around the expression of MAPK, Ki67, and CD31 in BTC in models To investigate the mechanism of tumor growth inhibition observed in BTC in models, tumor sections derived from xenografts were assessed for the expression of MAPK phosphorylation by IHC, and for Ki67 e CD31 expression by immunofluorescent analysis. As shown in Figure ?Determine3,3, the phosphorylation of MAPK was reduced in EGI-1 xenografts both by Panitumumab and Trametinib in monotherapy and.